Abstract Retroviruses induce a broad range of tumors in vertebrates. Although some retroviruses carry oncogenes in their genome, the vast majority of retroviruses do not encode such elements and thus, their capacity to induce tumors depends on integration near cellular proto-oncogenes. However, oncogene activation alone is not sufficient for tumor induction and additional events are required for tumor development. Murine Leukemia Virus (MuLV), which can spread as an oral and as a blood-borne pathogen, is highly proficient in causing leukemia. Intriguingly, tumor incidence within the same strain of infected mice varies between different facilities. Among environmental factors that may differ between the research labs, the variation in gut microbiota stands out.Thus, we sought to determine whether the microbiota contributes to virally-induced leukemogenesis. Accordingly, we monitored virally-induced leukemia in germ-free (GF, sterile) and specific pathogen free (SPF) conventionally raised BALB/cJ mice. Even though MuLV replication and spread were not affected in the absence of the microbiota, GF mice were significantly more resistant to the leukemia than SPF mice. Colonizing GF mice with a defined group of commensal bacteria (Altered Schaedler?s Flora), or with a single bacterium such as Lactobacillus murinus but not Parabacteroides distasonis or similar to E. coli and shigella (SECS) did not change virus replication but abolished the tumor-resistant phenotype of the colonized mice, indicating that some gut microbes have tumor-promoting properties. At the same time, GF mice lacking adaptive immune system (T and B lymphocytes) developed leukemia at a high rate suggesting that the gut microbes facilitate leukemia promotion by counteracting the adaptive immune response. Gene- expression analysis of the pre-leukemic spleens revealed several negative regulators induced by the microbiota. Among those, the ?V-set and Ig domain-containing 4? (VSig4) gene, a known negative regulator of T-cell responses attracted our interest because it was found upregulated in various human cancers. The gut microbiota has been implicated in both progression of cancers of colon and liver (?local? influence), as well as in systemic anti-cancer effect by enhancing the effect of anti-cancer immunotherapy. There is also a hint that the microbial environment can influence development of non-virally induced lymphomas. To identify the mechanism(s) by which the gut microbiota promotes leukemia, we will use our expertise in virology, mammalian genetics and gnotobiotic technologies (Dr. Golovkina, University of Chicago) and expertise in bacterial genetics and metabolomics (Dr. Fischbach, Stanford University). The goals of the proposal are: to determine the role of microbially regulated-VSig4 in virally-induced leukemogenesis; to characterize the bacterial compound(s) responsible for virally-induced leukemia development; to determine whether the gut microbiota influences leukemia development in a non-viral model of cancer.